1. Field of the Invention
The present invention relates to the field of metabolic engineering, and more particularly relates to a genetically engineered strain of Yarrowia lipolytica with enhanced extracellular secretion of α-ketoglutarate.
2. Description of the Related Art
As one of important intermediates in tricarboxylic acid cycle, α-ketoglutaric acid (α-KG) not only plays a key role in metabolic processes of microorganism, such as energy metabolism and synthesis of amino acids, proteins and vitamins, but also plays an important role in the regulation of Carbon/Nitrogen metabolic balance in microorganisms. It is important for study of regulation mechanism of nitrogen metabolism in microbes. As an essential intermediate in fine chemicals and pharmaceutical industry, α-KG is widely used in the synthesis of amino acids, vitamins and other small molecules, and has a wide range of applications in pharmaceutics, organic synthesis and nutritional supplement synthesis.
Due to the special role of α-KG in intracellular metabolism of microbes, when a large amount of α-KG is produced by screened strains during fermentation, it is unaviodable that a large amount of metabolism by-products, such as pyruvic acid, will accumulate in the final fermentation period. Short chain keto acids such as α-KG and pyruvic acid are weak electrolytes which exist in the form of neutral molecular or anion depending on the pH. As intercellular pH is higher than pKa of α-KG, α-KG and other organic acids predominately exist in their anion state. Excessive anions in cytoplasm result in the acidification of cytoplasm and interruption of cell metabolism. It is therefore necessary for α-KG in anion state to be transported cross cytoplasmic membranes by carboxylic acid transporter. However, when cells lack carbon source, carboxylic acid transporter will also need to transport specific carboxylates into cell as new carbon sources. Other carboxylic acids related to central metabolic pathway have similar secretion and absorption process. Therefore, the kinetic characteristics and regulation mechanism of specific carboxylic acid transporters located at cell membrane play a important role in regulating accumulation of carboxylic acids inside cells and in the fermentation broth.